Investigating host-microbiome interactions by droplet based microfluidics

Abstract

Background Despite the importance of the mucosal interface between microbiota and the host in gut homeostasis, little is known about the mechanisms of bacterial gut colonization, involving foraging for glycans produced by epithelial cells. The slow pace of progress toward understanding the underlying molecular mechanisms is largely due to the lack of efficient discovery tools, especially those targeting the uncultured fraction of the microbiota. Results Here, we introduce an ultra-high-throughput metagenomic approach based on droplet microfluidics, to screen fosmid libraries. Thousands of bacterial genomes can be covered in 1 h of work, with less than ten micrograms of substrate. Applied to the screening of the mucosal microbiota for β-N-acetylgalactosaminidase activity, this approach allowed the identification of pathways involved in the degradation of human gangliosides and milk oligosaccharides, the structural homologs of intestinal mucin glycans. These pathways, whose prevalence is associated with inflammatory bowel diseases, could be the result of horizontal gene transfers with Bacteroides species. Such pathways represent novel targets to study the microbiota-host interactions in the context of inflammatory bowel diseases, in which the integrity of the mucosal barrier is impaired. Conclusion By compartmentalizing experiments inside microfluidic droplets, this method speeds up and miniaturizes by several orders of magnitude the screening process compared to conventional approaches, to capture entire metabolic pathways from metagenomic libraries. The method is compatible with all types of (meta)genomic libraries, and employs a commercially available flow cytometer instead of a custom-made sorting system to detect intracellular or extracellular enzyme activities. This versatile and generic workflow will accelerate experimental exploration campaigns in functional metagenomics and holobiomics studies, to further decipher host-microbiota relationships.

The role of the microbiome in gastrointestinal inflammation

“…Over the past few decades, research related to the microbiome has intensified, facilitated by rapid advances in culture-independent, high-throughput genomic and metabolomic techniques [ 10–12 ]. Consequently, a greater understanding of microbiota population composition and host–microbe interactions has been achieved, especially in the context of human health and disease [ 11 , 13 , 14 ]. Whereas a balanced microbiota has been shown to play an important role in the maintenance of human health, impairment or imbalance in the makeup of the human microbiota (dysbiosis) can disrupt homoeostasis and lead to the onset or exacerbation of human disease [ 15 ].…”

Enrichment of gut microbiome strains for cultivation-free genome sequencing using droplet microfluidics

“…Last but not least, as the field aims to move from functional predictions to ecotypes of species, we require single-cell approaches ( Hatzenpichler et al., 2020 ) to combine genetic analysis with functional screens ( Berg et al., 2020 ). Droplet microfluidic methods allow the use of fluorescent probes indicating functional properties of cells as trigger for droplet sorting ( Xi et al., 2017 ) to enrich not only for the presence of genetic target sequences as shown here but also industrially relevant enzymatic activities ( Hosokawa et al., 2015 ; Tauzin et al., 2020 ; Zeng et al., 2020 ), cell-surface proteins via antibodies ( Cross et al., 2019 ), functionalized quantum dots ( Feng and Qian, 2018 ), or other probes targeting cell surfaces and bioactive content ( Terekhov et al., 2018 ). Such methods can also be extended to microfluidic chambers ( Leung et al., 2012 ).…”

“…Beyond the benefits of increased resolution through targeted experiments ( Cross et al., 2019 ; Grieb et al., 2020 ), microfluidic droplet screens are able to provide the required throughput ( Prakadan et al., 2017 ) to study complex ecosystems such as microbiomes ( Tauzin et al., 2020 ; Terekhov et al., 2018 ). Experiments in droplets can also substantially lower the amount of material and reagents used, cost, and contamination ( Hosic et al., 2016 ; Nishikawa et al., 2015 ) and provide additional information through functional enrichment ( Kintses et al., 2010 ; Tauzin et al., 2020 ; Terekhov et al., 2018 ; Yaginuma et al., 2019 ). An increasing diversity of microfluidic tools for bacterial single-cell analysis exists in the context of laboratory cultures.…”

FiberGrowth Pipeline: A Framework Toward Predicting Fiber-Specific Growth From Human Gut Bacteroidetes Genomes

“…Mucin is at a crossroad between dietary substrates and human secretion. Indeed, using droplet microfluidics, we recently demonstrated and characterized a new GH enzyme, active on human gangliosides, with similar structure to human mucin and milk oligosides and overrepresented in IBD patients' metagenomes (Tauzin et al, 2020). The mucin composition and degradation pathways still remain to be elucidated.…”

“…However, an accurate annotation of PUL seems difficult in metagenomics datasets. We already showed, using 40-kb E. coli fosmid libraries built from fecal samples or distal ileum mucosa, that glycosyl hydrolases were modular and subject to recent horizontal gene transfer, not just between phylogenetically close genomes but above the genus level (Tasse et al, 2010;Tauzin et al, 2020). A challenge remains to accurately annotate glycosyl hydrolase loci in metagenomic data, only feasible with long assembly with enough coverage.…”